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FATTY ACID PROFILE, CAROTENOID CONTENT, AND INVITRO ANTICANCER ACTIVITY OF KARIMUNJAWA AND

LAMPUNG SEA CUCUMBEREkowati Chasanah*1), Yusro Nuri Fawzya1, Kustiariyah Tarman*2),

Hedi Indra Januar1, and Muhammad Nursid1

1Research and Development Center for Marine. and Fisheries Product Competitiveness and Biotechnology,Jalan KS Tubun Petamburan VI, Jakarta, 10260, Indonesia;

2Faculty of Fisheries and Marine Sciences, Bogor Agricultural University, Jalan Raya Dramaga Kampus IPB,Bogor 16680, Indonesia

Abstract

Fatty acids and carotenoid has been known as an anticancer agent on both preventing and treating cancer disease.This study was conducted to analyze the fatty acid profile, carotenoid and in vitro anticancer activity of 12 seacucumber harvested from Karimunjawa and Lampung waters. The aim of the study was to determin the potency ofsea cucumbers as raw material for nutraceutical products. Fatty acid profile and carotenoid content werecharacterized by gas chromatography and spectrophotometry techniques, while in vitro anticancer activity wasassessed by MTT assay against cervix (HeLa), breast (T47D and MCF-7) and colon (WiDR) cancer cells. Resultsof the study showed polyunsaturated fatty acid (PUFA) dominated the composition of fatty acids in the samples fromboth locations. Holothuria sp. was detected to contain the highest amount of carotenoid. Furthermore, the highestin vitro anticancer activity was detected also in the sample of Holothuria sp. The activity of 30 ppm Holothuria sp.extract against HeLa cell was detected to be almost equal to the 5 ppm doxorubicin control. Concentration of 5 ppmHolothuria sp. extract also showed positive result in killing 50% of MCF-7 and T47D, but capable to 100% kill HeLaand WiDR cells. At concentration of 25 ppm, the extract was able to kill all the 4 cells tested. Statistical analysisshowed the amount of carotenoid and two particular fatty acid compounds (docosadienoic and eicosapentaenoicacid) significantly (P<0.05) contributed to the cytotoxic activity that was found in the sea cucumber samples. Thosecompounds were found in highest concentration from Holothuria sp harvested from Lampung waters, thus beingthe most prospective raw material for nutraceutical or functional food ingredient with anticancer potency.Keywords: anticancer, carotenoid, fatty acids, sea cucumber

Article history:Received: 9 September 2016; Revised: 28 October 2016; Accepted: 30 November 2016

Squalen Bull. of Mar. and Fish. Postharvest and Biotech. 11 (3) 2016, 117-124

www.bbp4b.litbang.kkp.go.id/squalen-bulletin

Squalen Bulletin of Marine and Fisheries Postharvest and Biotechnology ISSN: 2089-5690e-ISSN: 2406-9272

*Corresponding author.E-mail: ekowatichasanah@gmail.com

Copyright © 2016, Squalen BMFPB. Accreditation Number: 631/AU2/P2MI-LIPI/03/2015.DOI: http://dx.doi.org/10.15578/squalen.v11i3.269

1. Introduction

According to FAO statistics, Indonesia is thebiggest exporter of dried sea cucumber worldwide,and about 40–80 % are exported to China, Hong KongSAR, Japan, Republic of Korea, Taiwan, Singapore,Malaysia and Australia (Bordbar, Farooq & NazamidS, 2011). Well known as teripang, trepang, beche-de-mer, or gamat, sea cucumbers have been utilizednot only as food but also for traditional medicine inMiddle East and Asian countries, including Indonesia.Among the total number of existing sea cucumberspecies in the world, i.e about 1250 species (Bordbaret al., 2011), Darsono (2007) reported that there were

53 species of sea cucumber in Indonesia, and 29 ofthem are internationally traded which are mostlyHolothuriidae and Stichopodidae. In Indonesia,Lampung and Karimunjawa waters are locations wheresea cucumber sources has been harvested forcommercial dried sea cucumber product. Recently,a number of publications reported that some seacucumber species has been appl ied forpharmaceutical and cosmetical used, and has apromising future as important component for functionalfood products (Janakiram, Mohammed, & Rao, 2015;Ridhowati, Zakaria, Syah, & Chasanah, 2014). Manypublications report the potency of sea cucumber asantitumor agent and their possibility applications to

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the prevention and treatment of human diseases,including cancers (Janakiram et al., 2015; Nursid,Maharani, A.P., Riyanti, & Marraskuranto, E. 2016;Pérez-Espadas et al., 2014). However, exploration ofsea cucumbers originating from Lampung andKarimunjawa waters, particularly as anticancer agent,is still limited.

Fatty acid profile in sea cucumber is consideredunique. As bottom sediment feeder organism, seacucumber is considered healthy food that contains abranched chain fatty acid that has important role inrepairing tissue and other important role to keep hearthealthy and minimize cancer risk (Sicuro et al., 2012).The PUFA (poly unsaturated fatty acid) such as EPA(eicosa pentaenoic acid) and DHA (docosahexaenoicacid) which is usually abundant in deep fish is reportedto have anticancer activity both preventing and treatingthe cancer (Jing, Wu, & Lim, 2013; Vaughan, Hassing,& Lewandowski, 2013). Yoo et al. (2007) reported thatthree free fatty acids such as palmitic acid, (Z)-9-octadecenoic acid and octadecenoic acid fromProtaetia brevitarsis larva (PBL) are identified asbioactive compound against various cancer cell lines.Meanwhile Januar et al. (2014) reported that stearicacid was found to be an active substance isolatedfrom sea cucumber against cancer cell line MCF-7with IC50 of 10.32 ppm.

Tetraterpenoids or carotenoids is defined as organicpigments; in sea cucumber, they are obtained fromplants and algae, as well as several bacteria and fungithey consumed as debris. It is usually related well tothe biopotency of the sea cucumber such asantioxidant, antibacteria, and antitumor. Ibrahim (2012)reported that carotenoid in 3 Holothurian from Egyptianwaters had a role as antibacterial agent against S.aureus ATCC 6538, while Dang et al. (2007) reportedthat H. scabra, H. leucospilota and Stichopuschloronotus harvested from Malaysian waters werepotential sources of natural antioxidant and anticanceragents.

WHO (2014) reported that breast, colorectum andcervical cancer are the dominant cancer caused deathin Indonesia. For about 92,200 women deaths, 21.4%was caused by breast cancer, 10.3% by cervix utericancer and 9.5% by colorectum cancer. Therefore,many studies have great attention to find sources ofnatural products which have cytotoxic activity againstcancer. The objective of the research was to studyfatty acid and carotenoid profile of the sea cucumberfrom Karimunjawa and Lampung waters against 4 cellcancer lines.

2. Material and Methods

Sample collection and preservation. Live seacucumber were bought from Karimunjawa andLampung (Figure 1) fisherman on April 2013. Therewere 12 species of sea cucumbers (Figure 2)commonly caught and commercially marketed as driedsea cucumber in Karimunjawa and Lampung, the twoimportant sea cucumber sources in Indonesia. InKarimunjawa, the dominant sea cucumber marketedwas Stichopus, while from Lampung waters, the seacucumber caught and marketed were relatively varied.Out of 7 sea cucumber samples from Karimunjawawere 4 Stichopus sp. (K1, K2, K3, K6), Stichopusocellatus (K4), S. vastus (K5) and Actinopygalecanora (K7), while from Lampung waters, the 5 seacucumber samples were 2 Stichopus sp. (L1, L3), 1Actinopyga sp (L2) and 2 Holothuria sp. (L2, L4). Thesamples were divided into two parts. The first partwas taken for fatty acids profile, carotenoid content,and cytotoxic analysis. These samples were cleaned,degutted, and preserved at -80 °C under liquid nitrogen.The second part was taken for taxonomical analysis.Raw samples were chilled in coolbox using ice andtaxonomic analysis was conducted in Laboratory ofFisheries, Bogor Agricultural University based onmorphological characteristics, including dorsal andventral surface colors (were immediately recorded after

Figure 1. Sampling location (Lampung and Karimunjawa).

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cucumber extract was conducted using HeLa cells atextract concentration of 30 ppm. The highest cytotoxicextract was further tested on 4 tumor cell lines, i.eT47D (breast cancer), MCF7 (breast cancer), HeLa(cervix cancer), and WiDr (colon cancer) using serialdoses. A commercial cytotoxic agent (doxorubicin)was used as positive control to compare the level ofcytotoxicity. All analyses were conducted in duplicatesand the result was presented in average of theduplicate measurements.

Statistical Analysis. All data were log transformedto satisfy the requirement of normally distributed datain statistical multivariate analysis. The assessmentof data normality was conducted using the Shapiro-Wilk W test. Multivariate stepwise regression analysiswas employed to detect the relationship between fattyacid profile, carotenoid content, and in vitro anticanceractivity. Fatty acid components and carotenoid contentwere independent variables and cytotoxicity wasdependent variables. Statistical analyses wereconducted with Statistica Software v10.

3. Results and Discussion

3.1. Fatty Acids Profile

Fatty acid profiles of all sea cucumber studiedshowed that the l ipid was dominated bypolyunsaturated fatty acid (PUFA), both sea cucumberharvested from Lampung and Karimunjawa waters(Figure 3). The highest PUFA content was in L4 sample(Holothuria sp.) harvested from Lampung waters(Figure 3). Among PUFA, -linoleic acid; arachidicacid (18:3n6;20) was the highest (81.58 mg/100 g fresh

Figure 2. Sea cucumbers from Karimunjawa and Lampung waters.

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collection), tentacle type, number of calcareous rings,respiratory trees and gonads and types of spicules.

Fatty acid profile. Fatty acid was extracted fromthe fresh thawed sample according to Jensen (2008).The profile of fatty acids was analyzed by GasChromatography (GC) technique, with Flame IonizationDetector (FID) and Supelco 37 FAME Mix (CRM57885)fatty acids standard following to O’Fallon, Busboom,Nelson, & Gaskins, (2007).

Carotenoid analysis. Cleaned fresh 100 g sampleswere extracted with 250 mL ethanol. Extractionprocess was repeated twice and solvent removed underreduced pressure by rotary evaporator and freezedried. The analysis of carotenoid content had beenconducted by UV-Vis spectrophotometry assay. A five(5) mg dried extract was dissolved in 10 mL methanol(p.a.) and the total carotenoid was calculatedaccording to Lichtenthaler & Buschmann (2001)equation. The correction of blank absorption wasanalyzed before samples reading and automaticallycalculated in the instrument software during samplesanalyses.

Cytotoxicity against cell line. A five (5) mg driedextract was dissolved in 10 mL acetone. The processwas repeated two times. The acetone fractions wasdried by rotary evaporator and freeze dryer.Cytotoxicity test was performed using 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl-tetrazolium bromide(MTT) (Sigma) assay according to Ebada, Edrada,Lin, & Proksch, (2008). First screening of the sea

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meat, WB), fol lowed by cis-5,8,11,14,17Eicosapentanoic acid (20:3n3) EPA (10.528 mg/100g fresh meat), Arachidonis acid (20:3n3) (0.858 mg/100 g fresh meat) and 13,16-Docosadienoic acid(22:02) (0.728 mg/100 g fresh meat) and cis-11,14,17-Eicosatrienoic acid (20:3n3) (0.484 mg/100 g freshmeat). The highest EPA was detected in L5 (Stichopussp.) followed by L4 and L2 (Actynophyga sp.) (Table1). However, the sea cucumbers studied were poor ofdocosahexanoic acid (DHA).

Sea cucumber harvested from Lampung waterscontained more PUFA than those from Karimunjawawaters. Sea cucumber from Lampung waterscontained more arachidonic acid content, being thehighest were Stichopus sp. (L1) and Holothuria sp.(L4), while those from Karimunjawa contained less inthose two fatty acids. This study was in line withSvetashev, Levin, Cham, & Do, (1991) who reportedthat the major fatty acids discovered in almost allspecies of sea cucumber was PUFA. Sicuro et al.(2012) also reported that EPA was dominant in PUFAcontained in sea cucumber while this study found that Linoleic; Arachidic (18:3n6 ; 20) was dominat fattyacid among PUFA content. Svetashey et al. (1991)and Ridzwan et al. (2014) found that the dominantPUFA in their sea cucumber studied was arachidonicacid (-6 acid).

This study found SFA was dominant after PUFA(Figure 3), being the highest was in sample K4(Stichopus vastus) followed by sample L2 (Actinopygasp.) and K2 (Stichopus sp). Among SFA, palmiticacid was the dominant fatty acid. Study on seacucumber harvested from Kendari surrounding waters

(Fawzya, Januar, Susilowati, & Chasanah, 2015)reported that SFA was more dominant than PUFA andMUFA in the sea cucumber B. argus, H. fuscogilva,T. ananas. From this results, it can be inferred thatdifferent habitat, although from the same species,could produce sea cucumber with different fatty acidprofile, showing that waters and its environmental fromwhere the sea cucumber harvested has an importantrole in the chemical composition especially in thiscase was fatty acid profile. Postharvest handling andprocessing also influence the sea cucumber lipidquality, especially the PUFA which will very proneundergo oxidative changes, causing decreasing thePUFA content. Wen et al. (2010) reported that PUFAwas the least in dried sea cucumber and MUFA wasdominant in 8 dried sea cucumber followed by SFA.Chemical composition, nutritional status of the seacucumber including lipid and fatty acid profile was alsoinfluenced by animals feeding, physiologicalcharacteristics, life cycle and habitat of the specieswhere the sea cucumber has been harvested (Aidinet al., 2011; Neto et al., 2006; Vergara & Rodríguez,2016).

3.2. Carotenoid Content

Figure 4 showed that the carotenoid of the seacucumber from Lampung were 11 – 764 pg/g (WB).Meanwhile sea cucumber harvested from Karimunjawahad carotenoid of about 20-108 pg/g (WB). From thisdata, it can be suggested that sea cucumber fromLampung waters had higher total carotenoid thanthose from Karimunjawa waters. Among the seacucumber analyzed, L3 sample, Actinopyga sp. from

Figure 3. Fatty acid (mg/100 g fresh tissue) profile of sea cucumber from Karimunjawa (K) and Lampung (L) waters (L1, L2 : Stichopus sp.; L4: Holothuria sp.; L3, L5: Actinopyga sp.; K 1, 2,3, 6: Stichopus sp.; K4: Stichopus vastus; K5: Stichopus ocellatus; K7: Actinopyga lecanora).

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Table 1. PUFA profile of sea cucumber from Karimunjawa (K) and Lampung (L) waters (L1, L2: Stichopus sp.;L4 : Holothuria sp.; L3, L5 : Actinopyga sp.; K 1, 2,3, 6: Stichopus sp.; K4: Stichopus vastus; K5:Stichopus ocellatus; K7: Actinopyga lecanora)

Figure 4. Carotenoid content of sea cucumber Karimunjawa (K) and Lampung (L) waters (L1, L2 : Stichopus sp.; L4 : Holothuria sp.; L3, L5 : Actinopyga sp.; K 1, 2,3, 6 : Stichopus sp.; K4 : Stichopus vastus; K5 : Stichopus ocellatus; K7 : Actinopyga lecanora).

PUFA mg/100 g fresh meat L1 L2 L3 L4 L5 K1 K2 K3 K4 K5 K6 K7

Linolelaidic (18:2n6) 0 1.01 0.38 1.19 0 0 0.71 0 1 0 0 0

Linoleic (18:2n6) 0.59 1.71 0.62 1.06 0.39 0.39 1.5 0.85 1.81 0.64 0.47 0.61

gamma-Linoleic; Arachidic (18:3n6; 20)

33.64 28.85 28.67 81.58 22 12.53 32.02 32.43 42.3 24.91 8.25 15.22

alpha-Linoleic (18:3n3) 0.87 7.14 3.64 8.1 0.71 0 5.37 3.31 3.42 2.12 0 2.38

cis-5,8,11,14,17-Eicosapentaenoic (20:3n3)

4.83 6.19 3.87 10.53 2.94 1.51 2.64 0 0 0 0 0

cis-11,14,17-Eicosatrienoic; Behenic (20:3n6; 22)

0 0 0 0 0 0 0 0 0 0 0 0

cis-8,11,14,17-Eicosatrienoic (20:3n3)

0.68 0.71 0 0.48 0.43 0 0 0 0.75 0 0 0

Arachidonic (20:4n6) 1.06 0.51 0.63 0.86 0 0 0 0 0 0 0 0

cis-13,16-Docosadienoic (22:02)

0 0 0 0.73 0 0 0 0 0 0 0 0

cis-5,8,11,14,17-Eicosapentaenoic (EPA) (20:5n3)

0 0.3 0 0.51 2.49 0 0 0 0 0 0 0

 Total 41.67 46.43 37.8 105 28.96 14.43 42.25 36.58 49.28 27.67 8.72 18.21

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Figure 5. Cytotoxicity of sea cucumber extract Karimunjawa (K) and Lampung (L) waters (L1, L2: Stichopussp.; L4: Holothuria sp.; L3, L5: Actinopyga sp.; K 1, 2,3, 6: Stichopus sp.; K4: Stichopus vastus;K5: Stichopus ocellatus; K7: Actinopyga lecanora) against HeLa cell at concentration of 30 ppm;control used was doxorubicin in concentration of 5 ppm.

Figure 6. Dose response of Holothurian extract (L4) against four cancer cell lines.

Lampung surrounding waters, locally known asteripang kapuk or kelapa, had the lowest carotenoidcontent while sample L4 (Holothuria sp) was thehighest. This result may suggest the biopotency ofL4 samples. Carotenoid compound is the C40 skeletonof the parent tetraterpene that have been modified bychemical transformations such as oxidation or cyclization. Carotenoid visually can be detectedfrom the yellowish -reddish yelow color which absorbsvisible light at 400-450 nm.

3.3. In Vitro Anticancer Activity

Figure 5 showed the cytotoxicity result of seacucumber extract against HeLa cancer cell-line atconcentration of 30 ppm. Sea cucumber extract withcode L4, i.e Holothuria sp., showed the best cytotoxic

activity against HeLa cell, almost comparable withthe 5 ppm doxorubicin, a commercial anticancer agentused as control. This is an agreement with thecarotenoid data, where the highest carotenoid wasfrom Lampung sea cucumber L4 Holothuria sp.

Further study as showed in Figure 6 indicated thatthis Holothuria sp. extracts were active against 4cancer cell lines, i.e colon (WDr), cervix (HeLa), andbreast ( T47D and MCF-7). At low extract concentration(5 ppm), HeLa and WDr cells were more sensitive toL4 extract, suggesting that it was significantstatistically (p<0.05). While at higher concentration,the sensitivity toward L4 extract among the 4 testeddifferent cells were relatively similar (p>0.05); theextract could kill 100% all cells tested. It means thatin concentration of extract higher than 5 ppm, theextract contain relatively high active substances that

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was very toxic to all cells. From that Figure, it can beconcluded that the IC50 value will be reached whenthe dosage of 1-10 ppm extracts applied.

3.4. Relationship between Fatty Acids and Carotenoid Profile to Cytotoxicity

Multivariate stepwise regression analysis revealedthree variables (cis-13,16-Docosadienoic/DSA, cis-5,8,11,14,17-Eicosapentaenoic/EPA, and carotenoidcontent) significantly (P<0.05) contributed to the valueof cytotoxicity. Combination of these three variablesdetermined 89.22% (R2 = 0.89) to the value ofcytotoxic activity against HeLa cell lines. Partialbivariate regression analyses from these threevariables to cytotoxicity are shown in Figure 7. It isshown that combination of DSA (R2 = 0.88) andcarotenoid content (R2 = 0.84) was the major partialcontributor in cytotoxicity. Meanwhile, EPA contributedweakly, as the partial regression bivariate analysesonly showed weak determination value (R2 = 0.07).

These results may contrary to common finding thatomega-3 PUFA and omega-9 MUFA are the major fattyacids that play the most important role to keep hearthealthy and minimize cancer risk (Jing et al. 2013).PUFAs especially omega (n or )-3 fatty acids-eicosapentaenoic (20:5,n-3) (EPA) and

Figure 7. Partial bivariate regression between cis-13,16-Docosadienoic/DSA (a), cis-5,8,11,14,17-Eicosapentaenoic/EPA (b), and carotenoid (c), to sea cucumber cytotoxicity against HeLa celllines.

docosahexaenoic (22:6, n-3) acid (DHA) are believedto cause the lowering of the incidence of thrombosisand atherosclerosis. This study found other compound(cis-13,16-Docosadienoic) significantly enhance,comparing to EPA, the ability of extract against HeLacell lines. It is probable, specificity of cell lines thathad been used affect the results of cytotoxic activity.A particular cytotoxic compound may effects vary,against different cell lines. Other study in cytotoxicactivity of non-polar fraction from sea cucumber alsoshowed varied results. Nursid et al. (2016) reportedthat steroid was the active compound suspected insea cucumber Actinopyga sp. which its methanolicextract was able to inhibit WiDr and T47D cell linesviability with the LC50 value of 55.93 and 87.55 µg/ml,respectively. Further study on the fractionation andcytotoxicity evaluation of the Holothuria sp fromLampung waters found that the most active compoundwas stearic acid with IC50 of 10.32 ppm towards MCF-7 cells (Januar, Nursid, & Chasanah, 2014). Moreoverother fatty acid, such as arachidonic acid, has beenreported to take an important role for human growth,blood cloting and wound healing. Arachidonic acid isalso known as precursor of eicosapentanoic andprimary compound of cell membrane. In general, fattyacids and carotenoid are prospective anticanceragents. Therefore, sea cucumber, particularly the

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(a) (b)

(c)

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Holothuria sp harvested from Lampung waters in thisstudy, is the most prospective raw material fornutraceutical or functional food ingredient withanticancer potency.

4. Conclusion

This study support previous report that PUFA wasthe dominant fatty acid in the 12 sea cucumberstudied. Holothuria sp from Lampung contained thehighest PUFA among the sea cucumber studied. Ofthe 12 sea cucumber harvested from Lampung andKarimunjawa waters, the Stichopus sp (sample L4)also exhibited the highest cytotoxicity against 4cancer cell lines, i.e colon (WDr), cervix (HeLa), andbreast (T47D and MCF-7). At low extract concentration(5 ppm), HeLa and WDr cells were more sensitive toL4 extract. This cytotoxic ability against cancer celllines was supported by its carotenoid content.Combination of cis-13, 16 docosahexaenoic acid andcarotenoid was the major partial contributor of L4anticancer activity. These finding suggest thatHolothuria sp harvested from Lampung waters waspotential to be used as raw material for nutraceuticalproducts.

ReferencesAidin, M., Sevgili, H., Tufan, B., Emre, Y., & Köse, S. (2011).

Proximate composition and fatty acid profile of threediferent fresh and dried commercial sea cucumberfrom Turkey. Int. J. of Food Science and Technology46 : 500-508.

Bordbar, S., Farooq, A., & Nazamid, S. (2011). High-valuecomponents and bioactives from sea cucumbers forfunctional foods—A Review. Mar. Drugs, 9, 1761-1805.

Dang, N.H., Thanh, N.V., Kiem, P.V., Huong, M., Minh,C.V., & Kim,Y.H. (2007). Two new triterpeneglycosides from the Vietnamese sea cucumberHolothuria. Arch. Pharm. Res. 30 (11), 1387–1391.

Darsono, P. (2007). Teripang (Holothurioidea): Kekayaanalam dalam keragaman biota laut. J. Oseana XXXII(2): 1-10.

Ebada, E.S., Edrada, R.U., Lin, W., & Proksch, P. (2008).Methods for Isolation, purification and structuralelucidation of bioactive secondary metabolites frommarine invertebrates. Nat. Prod, 3(12): 1820-1831.

Fawzya, Y.N., Januar, H.I., Susilowati R., & Chasanah, E.(2015). Chemical composition and fatty acid profileof some Indonesia sea cucumbers. Squalen: Bulletinof Marine and Fisheries Postharvest andBiotechnology, 10(1): 27-34.

Ibrahim, H.A.H. (2012). Antibacterial carotenoids of threeHolothuria species in Hurghada, Egypt. The EgyptianJournal of Aquatic Research, (38), 185-194.

Janakiram, N.B., Mohammed, A., & Rao, C.V. (2015). Seacucumbers metabolites as potent anti-canceragents. Mar. Drugs 13 : 2909-2923.

Januar, H.I., Nursid, M., & Chasanah, E. (2014). Cytotoxicsaturated fatty acids from the Indonesian seacucumber Holothuria sp. Squalen Bulletin of Marine

& Fisheries Postharvest & Biotechnology, 9 (1), 11-15.

Jensen, S.K. (2008). Improved Bligh and Dyer extractionprocedure. Lipid Technol, 20(12):280-281.

Jing, K., Wu, T., & Lim, K. (2013). Omega-3polyunsaturated fatty acids and cancer. AnticancerAgents Med Chen, 13(8): 1162-77

Lichtenthaler, H.K., & Buschmann, C. (2001).Chlorophylls and Carotenoids: Measurement andCharacterization by UV-VIS Spectroscopy. CurrentProtocols in Food Analytical Chemistry F4.3.1-F4.3.8.John Wiley & Sons, Inc.

Neto, R.R., Wolff, G.A., Billet, D.S.M., Mackenzie, K.L., &Thompson, A. (2006). The influence of changing foodsupply on the lipid biochemistry of deepseaholothurians. Deep-sea Research 1, 53.

Nursid, M., Maharani, A.P., Riyanti, & Marraskuranto, E.(2016). Cytotoxic activity and secondary metabolitecharacteristics of sea cucumber Actinopyga sp.Methanolic extract. Squalen Bull. of Mar. and Fish.Postharvest and Biotech. 11 (1): 23-30.

O’Fallon, J.V., Busboom, J.R., Nelson, M.L., & Gaskins,C.T. (2007). A direct method for fatty acid methyl estersynthesis: Application to wet meat tissue, oils andfeedstuffs. J. Anim.Sci. 85: 1511-1521.

Pérez-Espadas, A.R., Verde-Star, M.J.,Rivas-Morales, C.,Oranday-Cárdenas, A.,Morales-Rubio, M.E., León-Deniz, L.V., Canul-Canché, J., & Quijano, L. (2014).In vitro cytotoxic activity of Isostichopus badionotus,asea cucumber from yucatan peninsula coast. Journalof Pharmacy and Nutrition Sciences, 4: 183-186.

Ridhowati, S., Zakaria, F., Syah, D., & Chasanah, E.(2014). Sea cucumber as anticancer agents and Itsdevelopment for functional food products. SqualenBulletin of Marine & Fisheries Postharvest &Biotechnology, 9 (2): 85-96.

Ridzwan, B.H., Hanita, M.H., Nurzafirah, M.,Norshuhadaa, M.P.S., & Hanis, Z.F. (2014). Free fattyacids composition in lipid extracts of several seacucumbers species from Malaysia. InternationalJournal of Bioscience, Biochemistry andBioinformatics, 4(3): 204-207.

Svetashev, V.L., Levin, V.S., Cham, N.L., & Do, T.N. (1991).“Lipid and fatty acid composition of Holothurians fromtropical and temperate waters,” Comp. Biochem.Physiol., vol. 4, pp. 489-494, 1991.

Sicuro, B., Piccino, M., Gai, F., Abete MC., Danieli A., DapraF., Mioletti S., & Vilella, S. (2012). Food quality andsafety of Mediterranean sea cucumbers Holothuriatubulosa dan Holothuria polii in Southern Adriatic Sea.Asian J. Anim. Vet. Adv. : 1-9.

Vergara, W., & Rodríguez, A. (2016). Nutritionalcomposition of sea cucumber Isostichopus sp.Natural Resources, 7: 130-137.

Vaughan, V.C, Hassing, M-R., & Lewandowski, P. A.(2013). Marine polyunsaturated fatty acids and cancertherapy. British Journal of Cancer (2013) 108, 486–492.

Yoo, YC1., Shin, B.H., Hong, J.H., Lee, J., Chee, H.Y.,Song, K.S., Lee, K.B. (2007). Isolation of fatty acidswith anticancer activity from Protaetia brevitarsis larva.Arch.Pharm.Res. 30(3): 361-365.

Wen, J1., Hu, C., & Fan, S. (2010). Chemical compositionand nutritional quality of sea cucumbers.J. Sci FoodAgic 0(14): 2469-74.

WHO. 2014. Cancer country profiles 2014-Indonesia.

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